We have previously shown that DNA sequences located within the simian virus 40 (SV40) GC-rich, 21-bp repeats constitute an important transcriptional control element of the SV40 late promoter. To gain further insight into the mechanics by which the SV40 GC-rich repeats function, we have analyzed the transcriptional properties of several recombinant DNAs. These results suggest that the SV40 GC-rich sequences can function as independent RNA polymerase II transcriptional-control elements. In vitro competition studies demonstrated that sequences within the GC-rich 21-bp repeats, in the absence of either the SV40 early or late -25 transcriptional control signals (TATA box) or the major RNA initiation sites, efficiently competed for transcription factors required for SV40 early and late RNA synthesis. Our transcription studies also demonstrated that in the absence of contiguous SV40 transcription control sequences, these GC-rich sequences stimulated initiation of bidirectional transcription from proximally located sequences. We further demonstrated that the 21-bp repeat region can stimulate in vitro transcription from the heterologous adenovirus 2 major late promoter. We have also analyzed the transcriptional properties of a series of mutant templates which contain DNA insertions between the SV40 21-bp tandem repeats and the early Goldberg-Hogness TATA box. Our results suggest that the SV40 early G-H sequences require the presence, within a fixed distance, of upstream regulatory elements in order to function efficiently. When this interaction is disrupted by inserts of increasing distance, SV40 early RNA initiation sites are switched upstream to the "late-early" RNA initiation sites.